Neuroscience Program, Dominican University, River Forest, Illinois, United States of America.

Abstract

We used Aplysia californica to compare the transcriptional changes evoked by long-term sensitization training and by a treatment meant to mimic this training, in vivo exposure to serotonin. We focused on 5 candidate plasticity genes which are rapidly up-regulated in the Aplysia genus by in vivo serotonin treatment, but which have not yet been tested for regulation during sensitization: CREB1, matrilin, antistasin, eIF3e, and BAT1 homolog. CREB1 was rapidly up-regulated by both treatments, but the regulation following training was transient, falling back to control levels 24 hours after training. This suggests some caution in interpreting the proposed role of CREB1 in consolidating long-term sensitization memory. Both matrilin and eIF3e were up-regulated by in vivo serotonin but not by long-term sensitization training. This suggests that in vivo serotonin may produce generalized transcriptional effects that are not specific to long-term sensitization learning. Finally, neither treatment produced regulation of antistasin or BAT1 homolog, transcripts regulated by in vivo serotonin in the closely related Aplysia kurodai. This suggests either that these transcripts are not regulated by experience, or that transcriptional mechanisms of memory may vary within the Aplysia genus.

A. Long-term sensitization training protocol. Training consisted of 4 rounds of shock (30 minute interval). In each round, a 10 s shock (90mA AC, 0.5 s on, 0.5 s off) was applied to one side of the body. Pleural ganglia from the trained and untrained side were harvested separately 1 or 24 hours after training ended for qPCR analysis. In the 24-hour group, T-SWR duration was characterized before (pre-test) and 24 hours after (post-test) training. B. Mean T-SWR durations (±1 SEM) before and 24 hours after long-term sensitization in the 24 hour group (n = 14). T-SWRs were evoked via weak electrical shock to implanted electrodes in the tail and measured from the time of siphon contraction to the first sign of siphon relaxation. For each animal, pre-test and post-test responding was measured on the trained and untrained side separately as the mean of 3 T-SWRs. The p value shown is for a paired t-test comparing pre-test and post-test responses on the trained side. The comparison on the untrained side was not significant. C. Mean transcriptional changes (± 1SEM) 1 and 24 hours after long-term sensitization training (ns = 10, 13 respectively except for 1-hour C/EBP where n = 11). Fold changes are calculated as the ratio of transcript from the trained side to the untrained side. Data are shown on a log scale, and the dotted line at 1 indicates no change (equal levels of transcript in the treated and control animal). * Indicates the mean-fold change is significantly different than 1 by a one-sample t-test (p<0.05).

A. Protocol for in vivo 5-HT exposure. Experimental animals were immersed in artificial sea water (ASW) with 250 µM 5-HT for 2 hours; pleural ganglia were harvested for qPCR immediately afterwards. Each treated animal was matched with a control animal processed at the same time but immersed in ASW without 5-HT. To ensure this protocol produces long-term sensitization, a parallel behavioral experiment was conducted in which T-SWR durations were measured before (pre-test) and 24 hours after (post-test) treatment with either in vivo 5-HT or ASW. B. Mean T-SWR durations (±1 SEM) before and 24 hours after control ASW (n = 6) or in vivo 5-HT exposure (n = 8). T-SWRs were evoked via weak electrical shock to implanted electrodes in the tail and measured from the time of siphon contraction to the first sign of siphon relaxation. For each animal, pre-test and post-test responding was measured as the mean of 6 T-SWRs alternating between the left and right sides at a 5 minute ISI. The p value shown is for a paired t-test comparing pre-test and post-test responses within the treated group. The same comparison within the control ASW group was not significant. C. Mean transcriptional changes (± 1SEM) following in vivo 5-HT exposure (n = 10 pairs). Fold changes are calculated as the ratio of transcript in each treated animal versus its matched control. Data are shown on a log scale, and the dotted line at 1 indicates no change (equal levels of transcript in the treated and control animal). * Indicates the mean fold-change is significantly different than 1 by a one-sample t-test (p<0.05).